Quantitation of glycated hemoglobins in human adult blood by capillary isoelectric focusing

Electrophoresis
M ContiP G Righetti

Abstract

A precise and reproducible method for assessment of glycated hemoglobin in human adult red blood cells is reported, based on capillary isoelectric focusing (IEF). In order to obtain baseline resolution between adult hemoglobin (Hb A) and its glycated form (Hb A1c), two species which differ by minute delta pI values, < 0.03 pH units, the following procedure was adopted: the focusing mixture consisted of 5% Ampholine, pH 6-8, 0.5% Pharmalyte, pH 3-10, 3% short-chain liquid polyacrylamide and an equimolar mixture of two "separators", 0.33 M beta-alanine and 0.33 M 6-aminocaproic acid. The last two compounds flatten the pH gradient in the pI region of the two Hbs, thus allowing full separation. Additionally, the Hb samples, instead of being pulse-loaded, are uniformly distributed in the background electrolyte. A longer capillary life-time is obtained if all nonbuffering ions are eliminated; thus, as catholyte, 50 mM Lys (pH 9.7) is utilized and as anolyte 50 mM acetic acid (pH 3.5) is adopted. The percentages of Hb A1c, as obtained by capillary IEF, are in good agreement (+/- 6%) with data obtained by one of the standard zone electrophoretic methods in clinical chemistry, i.e., the Helena REP Glyco gel system.

References

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Citations

Feb 10, 1998·Electrophoresis·R Rodriguez-DiazM Zhu
Feb 10, 1998·Electrophoresis·F von Heeren, W Thormann
Apr 5, 2011·Analytical and Bioanalytical Chemistry·Lauren M RamsayNorman J Dovichi
Jun 25, 2004·Journal of Chromatography. a·Pier Giorgio Righetti
Jan 19, 1999·Journal of Chromatography. B, Biomedical Sciences and Applications·M A Jenkins
Jan 30, 2003·Journal of Chromatography. B, Analytical Technologies in the Biomedical and Life Sciences·Alessandra Vincenzi Jager, Marina Franco Maggi Tavares
Jul 26, 2003·Clinical Chemistry and Laboratory Medicine : CCLM·Margaret Jenkins, Sujiva Ratnaike
Dec 16, 1998·Electrophoresis·S Suzuki, S Honda
Oct 23, 2012·Electrophoresis·Csilla PágerFerenc Kilár
Nov 28, 2012·Proteomics·Pier Giorgio RighettiAttilio Citterio
Apr 30, 2002·FASEB Journal : Official Publication of the Federation of American Societies for Experimental Biology·Fortunata BaroneAntonio Filippini
Jan 13, 2018·Analytical and Bioanalytical Chemistry·Ji-Cheng NiuZhi-Yong Wu

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